Methods of using primer molecules for enhancing the mechanical performance of tissue adhesives and sealants

ABSTRACT

The present invention concerns novel methods for enhancing the mechanical performance of tissue adhesives and sealants which comprises employing a primer molecule in association with a tissue adhesive or sealant, wherein the primer molecule serves to enhance the strength of the interface between the tissue and the adhesive matrix. The primer molecules described herein function to interact with a protein present in the tissue, thereby altering its characteristics to make it more amenable to bonding with the adhesive matrix. Primer molecules may be applied to the tissue independently from the tissue adhesive or sealant or may be mixed with the tissue adhesive or sealant prior to application to the tissue.

FIELD OF THE INVENTION

The present invention is directed to generally applicable methods forusing primer molecules to enhance the adhesion of tissue adhesive ortissue sealant compositions to tissues.

BACKGROUND OF THE INVENTION

In many situations, there is a need to bond separated tissues or to sealdefects in tissues. Sutures and staples are effective and wellestablished wound and tissue defect closure devices. However, there aresurgical techniques where classical repair procedures areunsatisfactory, limited to highly trained specialists (e.g.,microsurgery), or not applicable due to tissue or organ fragility,inaccessibility (e.g., endoscopy procedures), or loss of gases orfluids, including capillary “weeping”. Tissue adhesives and sealantshave been developed to meet these needs. They may be used to seal orreinforce wounds that have been sutured or stapled, as well as findingindependent use. The leading commercial products are fibrin glues andcyanoacrylates. However, both products have significant limitationswhich have prevented their widespread use.

In this regard, one of the major limitations encountered in thedevelopment and/or use of tissue adhesive and sealant compositions istheir inability to form a sufficiently strong bond to tissues.Therefore, tissue adhesives and sealants may have to be employed incombination with sutures and/or staples so as to reduce the tissuebonding strength required for acceptable performance. As describedabove, however, there are many indications where the use of suturesand/or staples is undesirable, inappropriate or impossible.

As demonstrated by the Applicants herein, mechanical failure of bondsformed by tissue adhesive and sealant systems may occur at the interfacebetween the crosslinked adhesive matrix and the tissue. The inability ofthe adhesive matrix to form a strong interface or bond with tissues ismost likely due to the fact that various proteins in the tissue are notreadily amenable to non-covalent and/or covalent interactions with thetissue adhesive or sealant components as applied and/or during and aftercuring. For example, collagen present in tissues is a highly aggregated,insoluble protein which, because of its physical characteristics, is notreadily amenable to interacting with tissue adhesive or sealantcomponents. The same often holds true for other tissue-associatedproteins such as actin and myosin. As a result, for most tissues andadhesive and sealant systems, failures are generally believed to occurat the interface between the crosslinked adhesive matrix and one or moretissue-associated protein such as collagen, actin and myosin.

One possibility for improving the mechanical performance of a tissueadhesive or sealant is to strengthen the interface between atissue-associated protein and the adhesive matrix by altering thephysical characteristics of the adhesive or sealant components to moreclosely approximate those of protein components of the tissue, therebymaking the two components more compatible and amenable to non-covalentand/or covalent interaction. As one example, bonding of a dentalrestoration to the dentine of a tooth requires the establishment ofadhesion between the demineralized collagen present in the dentine and amethacrylate adhesive matrix. In one study, alteration of the vinylmonomers present in a methacrylate adhesive matrix to more closely matchthe solubility parameter of collagen present in the dentine resulted inan increased strength of bonding of a dental restoration. (Miller,Adhesive Bonding to Dentin with Isocyanate Copolymers”, Ph.D.Dissertation, University of Missouri—Columbia (1995)). However, thedevelopment of physiologically acceptable adhesives and sealants whichclosely match the characteristics of native collagen has, for the mostpart, been unsuccessful.

There is, therefore, a need for novel methods for enhancing the adhesionof adhesive or sealant compositions to tissues. More specifically, thereis a need for novel methods of strengthening the adhesivematrix/tissue-associated protein interface so as to enhance themechanical properties of bonds created by tissue adhesives and sealants,wherein those methods are generally applicable to a variety of differenttissues and adhesive and sealant systems.

SUMMARY OF THE INVENTION

The present invention provides generally applicable methods forenhancing the mechanical performance of adhesive and sealant systems foruse with both soft and hard tissues in vivo. The methods of the presentinvention are based at least in part upon the demonstration that certain“primer molecules” are capable of interacting with native component(s),particularly protein(s), in tissue, thereby altering the physicalcharacteristics of those components(s) to better match thecharacteristics of the crosslinked matrix formed by the tissue adhesiveor sealant components as applied and/or during and after curing. Bycausing the physical characteristics of tissue-associated components tobetter match those of the adhesive or sealant components, one enhancesthe ability of a tissue adhesive or sealant of interest to form astronger adhesive matrix/tissue-associated component interface which, inturn, results in stronger attachment of the adhesive matrix to thetissue. The primer molecules described herein are useful with virtuallyall adhesives or sealants for soft and hard tissues in which thestrength of the bond formed thereby is limited by failure at theinterface between the adhesive matrix and the tissue.

In accordance with the present invention, novel methods for enhancingthe mechanical performance of tissue adhesives and sealants areprovided. In one embodiment, the present invention is directed tomethods for maintaining separated tissues in proximate relationshipwhich comprise applying to the separated tissues (a) a primer moleculeand (b) a tissue adhesive, wherein the primer molecule and the tissueadhesive are applied in an amount effective to maintain the separatedtissues in proximate relationship when they are brought into such arelationship. In another embodiment, methods for sealing a defect in atissue are provided, wherein the methods comprise applying to the defect(a) a primer molecule and (b) a tissue sealant, wherein the primermolecule and the tissue sealant are applied in an amount effective toseal the defect in the tissue. The primer molecules are such that theyare capable of physically interacting with one or more proteins presentin the tissue, thereby allowing the tissue to form a strong bondedinterface with the crosslinked tissue adhesive or sealant (i.e.,adhesive matrix). In certain embodiments, the primer molecule may becapable of physically and/or covalently interacting with collagen, actinand/or myosin present in the tissue. The herein described methods arecapable of maintaining separated tissue in proximate relationship orsealing a defect in tissue with greater bond strength than when thetissue adhesive or sealant is employed in the absence of a primermolecule. Primer molecules which find use herein include, for example,chaotropic agents, dyes which are capable of staining tissue-associatedproteins such as collagen, actin or myosin, proteins, oligopeptides, andthe like.

The present invention is also directed to compositions and kits whichare useful for maintaining separated tissue in a proximate relationshipor sealing a defect in tissue and which comprise a tissue adhesive orsealant and a primer molecule which is capable of physically interactingwith a protein present in the tissue, thereby rendering the tissuecapable of forming a bonded interface with the crosslinked matrix of thetissue adhesive or sealant.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to generally applicable methods forenhancing the mechanical performance of adhesives and sealants for usewith hard and soft tissues, wherein those tissues may be associated witha living organism or otherwise. The methods of the present invention arebased at least in part on the demonstration that certain “primermolecules” are capable of interacting with one or more native componentsin tissue, thereby altering the physical properties of that component tobetter match the characteristics of the adhesive or sealant componentsas applied and/or during and after curing. In a preferred embodiment,the tissue component is a protein. Because the protein in the tissuewill then possess physical properties (in terms of wettability,swellability, structure, etc.) which enhance the ability of the adhesiveor sealant components to non-covalently and/or covalently interact withthe altered protein, thereby forming a stronger adhesivematrix/tissue-associated protein interface, a stronger bond of theadhesive matrix to the tissue results. In various preferred embodiments,the tissue-associated protein that interacts with the primer molecule iscollagen, actin or myosin.

In this regard, achieving intimate contact between adhesive componentsand an adherend is required for increasing the strength of adhesivejoints (Kaelble, “Physical Chemistry of Adhesion”, Wiley-Interscience,New York (1971) and Iyengar et al., Journal of Applied Polymer Science11:2311-2324 (1967)). However, wetting and intimate contact of tissueadhesive or sealant components with tissue-associated proteins is oftenimpeded by the insolubility and high degree of aggregation of many ofthe proteins present in the tissue. Applicants demonstrate herein thatprimer molecules which interact with tissue-associated proteins arecapable of making those proteins more accessible to interaction with thecomponents of a variety of different tissue adhesives and sealantswhich, in turn, enhances the mechanical performance of these tissueadhesives and sealants.

As such, the term “primer molecule”, “primer” and grammaticalequivalents thereof as used herein refer generally to molecules whichare capable of physically interacting with one or more native proteinspresent in a tissue and altering at least one of its native physicalcharacteristics, thereby rendering the tissue capable of forming abonded interface with a tissue adhesive or sealant matrix. By“physically interacting” is meant that the primer molecule comes intoactual physical contact with the protein present in the tissue, therebyaltering one or more of its native properties. By rendering the tissue“capable of forming a bonded interface” with a tissue adhesive orsealant is meant that after the tissue-associated protein physicallyinteracts with the primer molecule, the tissue is then capable ofnon-covalently or covalently interacting with the tissue adhesive orsealant components so as to strengthen the interface between the tissueand the crosslinked adhesive matrix. This phrase is intended toencompass situations where the tissue is initially incapable ofinteracting with the adhesive or sealant components as well assituations where the primer enhances the tissue's initial capability ofinteracting with the adhesive or sealant components. Thus, a “bondedinterface” between a crosslinked adhesive matrix and the tissue may be aresult of non-covalent and/or covalent interactions between thecrosslinked adhesive matrix and the tissue. Tissue-associated proteinsthat interact with primer molecules of the present invention include,for example, collagen, actin, myosin, and the like.

In one embodiment, primer molecules which find use in the presentinvention are capable of altering the degree of hydrogen bonding ofcollagen molecules to each other, altering the electrostatic orhydrophobic nature of the collagen and/or its degree of hydration,altering its density and/or degree of openness in its structuralorganization and/or altering its degree of association with other tissuecomponents, thereby rendering it more wettable, swellable orstructurally “open”. The increased wettablility, swellability and/or“openness” of the collagen in the tissue then enhances the tissue'scompatibility towards the components of the tissue adhesive or sealantand the ability to non-covalently and/or covalently interact with anadhesive matrix, thereby resulting in a stronger bonded interface.“Stronger bonds” are generally greater than about 1.3-fold stronger thanbonds formed by a tissue adhesive or sealant in the absence of a primermolecule in a lap shear tensile test, usually from about 1.3- to 10-foldstronger, more usually from about 1.3-fold to 8-fold stronger,preferably from about 2- to 8-fold stronger and more preferably fromabout 2.5- to 6-fold stronger.

Primer molecules which find use in the methods of the present inventionare numerous and come from diverse classes of different compounds, allof which, however, are capable of interacting with a native protein in atissue, thereby altering at least one of its native physicalcharacteristics so as to render the tissue more compatible for forming abonded interface with an adhesive matrix. For example, primer moleculeswhich find use herein include aqueous chaotropic agents such as urea,guanadinium chloride and phosphatidyl choline. Such “chaotropic agents”are compounds known to be capable of disrupting hydrogen bonding in,solubilizing and/or denaturing proteins, including highly orderedproteins such as collagen, thereby rendering those proteins moreaccessible to interact with the components of a tissue adhesive orsealant which will become crosslinked to form an adhesive matrix.

Other primer molecules which are capable of interacting with nativetissue proteins to alter their physical characteristics and, therefore,which find use in the presently described methods include dyes which arecapable of staining various tissue components, including collagen, actinand myosin. Examples of such dyes are numerous and well known in theliterature directed to histology and the technology of dying leather,cellulose, wood, etc. including, for example, Lille, ed., “Conn'sBiological Stains”, 9th ed., Williams & Wilkins, Baltimore, Md. (1977),Clark, “Staining Procedures”, 4th ed., Williams & Wilkins, Baltimore,Md. (1981) and Green, “The Sigma-Aldrich Handbook of Stains, Dyes, &Indicators”, Aldrich Chemical Company, Inc., Milwaukee, Wis. (1990),each of which is expressly incorporated herein by reference.

In preferred embodiments, the dyes which are employed in the presentlydescribed methods are sulfonated aromatic compounds or xanthenes. Onerelated sulfonated aromatic compound, oligo(1-naphthalenesulfonate-co-formaldehyde) has previously been shown to specificallyinteract with bovine hide collagen (Takata et al., Hikaku Kagaku33(3):151-156 (1987) and Chemical Abstracts 108:206608m (1987)).Sulfonated aromatic dyes which find use as primer molecules include, forexample (where the appropriate catalogue numbers from the “CatalogHandbook of Fine Chemicals 1996-1997”, Aldrich Chemical Company, Inc.,Milwaukee, Wis., are shown in parentheses), Brilliant Blue G (Aldrich20,140-5), Evan's Blue (Aldrich 20,633-4), Chicago Sky Blue 6B (Aldrich20,138-3), Cibacron Blue 3GA (Sigma C9534), Cibacron Brilliant Yellow3G-P (Aldrich 22,847-8), Brilliant Blue R (Aldrich 20,140-5), LissamineGreen B (Aldrich 19,958-3), Acid Blue 92 (Aldrich 21,042-0), CibacronBrilliant Red 3B-A (Aldrich 22,845-1), Acid Red 97 (Aldrich 21,039-0),Trypan Blue (Aldrich 30,264-3), New Coccine (Aldrich 19,973-7), Orange G(Aldrich 86,128-6), Hydroxy Napthol Blue di-sodium salt (Aldrich21,991-6), Ponceau S (Aldrich 14,119-4), Bordeaux R (Aldrich 20,962-7),Aniline Blue (Aldrich 41, 504-9), Reactive Black 5 (Aldrich 30,645-2),and the like. Xanthene dyes include Eosin Y (Aldrich 11,983-0), Eosin B(Aldrich 86,100-6), Erythrosin B (Aldrich 19,826-9), Rose Bengal(Aldrich 19,825-0), and the like. Sulfonated aromatic compounds andxanthenes which find use may also be devoid of a chromophore in thevisible range of wavelengths as long as they function to physicallyinteract with a tissue-associated component such as collagen, actin ormyosin. Colors may be removed, especially from the triarylmethyl dyes,via reduction to their leuco-forms using well known techniques. Theanalogs to azo dyes may be synthesized by means of substituting1,2-ethanediyl groups for azo linkages present in the colored molecules,also by well known techniques.

In addition to the aromatic sulfonate compounds described above, othercompounds which are capable of interacting with tissue-associatedproteins and which will find use herein include, for example, aliphaticsulfonates, aromatic or aliphatic sulfates, phosphates and carboxylates,and oligomers and polymers thereof, all of which are commerciallyavailable or the synthesis of which is well within the skill level inthe art.

Other primer molecules which are capable of interacting with nativetissue-associated proteins to alter their physical characteristics and,therefore, which find use in the presently described methods include,for example, mammalian proteins such as fibrinogen, fibronectin andalbumin, as well as recombinantly-produced repetitive unit proteinpolymers such as SELP0K and SELP0K-CS1, wherein the synthesis of therecombinantly-produced repetitive unit protein polymers is described indetail in U.S. Pat. No. 5,243,038, PCT/US89/05016, PCT/US92/09485 andPCT/US96/06229, the disclosures of which are expressly incorporated byreference in their entirety. Such proteins are capable of interactingwith tissue-associated proteins as they exist in native tissue andaltering at least one of the physical characteristics thereof, therebyallowing the components of a tissue adhesive or sealant to more readilyinteract with the tissue to form a stronger bonded interface between thecrosslinked adhesive matrix and the tissue.

Primer molecules may also comprise oligomeric structures such asoligopeptides, oligonucleotides, oligosaccharides, and the like, as longas they are capable of physically interacting with a tissue-associatedprotein. For example, oligomeric combinatorial libraries of peptides,nucleotides or saccharides may by prepared using well known chemical orbiological combinatorial synthesis techniques and may be screened forthe presence of members which function to physically interact with atissue-associated protein such as collagen, actin, myosin, and the like.See, for example, Owens and Baralle, EMBO J. 5(11):2825-2830 (1986).Library members which are identified as being capable of interactingwith a tissue-associated protein may be screened in lap shear tensilestrength assays to identify those which are useful as primer moleculesin the presently described methods. Any experimentation required toidentify such primer molecules is routine and well known in the art.

The primer molecules which find use in the present invention inherentlyinteract with one or more tissue-associated protein and may also bemodified to interact strongly with components of the adhesive matrixitself, thereby effectively forming a “bridge” between the protein ofthe tissue and the crosslinked adhesive matrix. For example, acrylate,methacrylate, acrylamide and methacrylamide decorated primer moleculeswill be effective in improving the interaction between vinyl-basedadhesives or sealants and tissue proteins, wherein the interactions maybe both ionic and covalent. Additionally, primer molecules which finduse herein may be employed either in monomeric or oligomeric, preferablylower oligomeric, form. Such primer molecules are intended to beencompassed by the present invention.

In addition to the above referenced primer molecules, other primermolecules which find use in the presently described methods may bereadily identified without undue experimentation on the part of theordinarily skilled artisan. For example, as disclosed herein, primermolecules which find use in the presently described methods are thosecompounds which are capable of physically interacting withtissue-associated proteins. Assays designed to screen large numbers ofpotentiol compounds for their ability to bind to and/or otherwiseinteract with native tissue proteins are well known in the art and/ormay be routinely devised by those skilled in the art. For example,potential primer molecules which possess a chromophore (such as a dye)or which are modified to possess a chromophore can be screened for theirability to stain either fibrillar collagen or skin coupons. Moreover,such substrates can be treated with potential primer molecules, whereinthose that are capable of physically interacting with proteins in thetissue would be expected to result in a substrate that is more highlysolvated with water or that displays an altered collagen meltingtemperature (Nothbohm et al., J. Prot. Chem. 11:635-643 (1 992) andGrade et al., J. Biomed. Matr. Res. 25:799-811 (1991)). Such assays maybe routinely employed to identify compounds which are capable ofphysically interacting with native tissue proteins as they exist intissue.

Compounds identified as being capable of physically interacting with oneor more tissue-associated proteins may then be employed in combinationwith a tissue adhesive or sealant of interest to determine if thosecompounds are capable of enhancing the mechanical performance of thetissue adhesive matrix, either by increasing the mechanical strength ofthe tissue bond and/or by increasing the time that the bond is capableof holding the separated tissue in proximity. Lap shear tensile testingassays designed to test the ability of a molecule to enhance themechanical performance of a tissue adhesive or sealant are described indetail below. The use of these assays to identify new primer moleculesis routine and does not require undue experimentation on the part of theordinarily skilled artisan.

The primer molecules described herein may be employed in methods forenhancing the mechanical performance of a wide variety of differenttissue adhesive and sealant systems. In fact, the performance ofvirtually any tissue adhesive or sealant system, either presently knownor later developed, which is capable of bonding to any anatomical sitecan benefit from improved contact between the adhesive matrix componentsand the tissue, and will show enhanced performance if the bond strengthof that tissue adhesive or sealant is limited by failure at theinterface between the tissue and the adhesive matrix. Such tissueadhesive or sealant systems include the well known methacrylate systems,and the like. Adhesive matrices comprising synthetic polymers such aspolyethylene glycol, polyvinylalcohols, polyesters, and the like,including derivatives may also find use in combination with a suitablecrosslinking agent. In one embodiment, the adhesive matrix of the tissueadhesive or sealant employed may also comprise either natural orrecombinantly-produced protein or combinations thereof, wherein thoseproteins are capable of being crosslinked by a suitable crosslinkingagent to form an adhesive matrix. Natural proteins which find use forincorporation into a tissue adhesive or sealant include, for example,gelatin (with or without resorcinol), fibrin, fibrinogen, albumin,casein, silk fibroin, keratin, mussel adhesive protein, collagen,succinylated gelatin derivatized with cystein, as well as a variety ofother well known natural proteins that are useful for adhesive orsealant purposes.

In addition to the natural proteins described above, variousrecombinantly-produced proteins may also find use in tissue adhesivesand sealants. Not only may the natural proteins described above berecombinantly produced, but also various crosslinkable non-naturalrecombinant proteins will find use herein. Preferred non-naturalrecombinantly produced proteins include proteins which compriserepeating units of naturally occurring amino acid sequence blocks fromsuch naturally occurring structural proteins as fibroin, elastin,collagen, keratin, and the like. The recombinant preparation of thesenon-natural repetitive unit proteins is described in detail in U.S. Pat.No. 5,243,038, PCT/US89/05016, PCT/US92/09485 and PCT/US96/06229 (thedisclosures of which are expressly incorporated herein by reference intheir entirety), wherein the adhesive and crosslinking properties ofthese proteins are also discussed. Preferred repetitive unit proteinsfor use in the presently described methods include SELP8K, SELP0K-CS1and SELP0K, whose preparation and amino acid sequences are described inPCT/US96/06229.

The proteins described above will be capable of being crosslinked toform an adhesive matrix. For crosslinking purposes, these proteins willcomprise one or more reactive functionalities, generally present on oneor more amino acids of the protein, and may include such functionalitiesas amino, e.g., lysine, carboxyl, e.g., aspartate and glutamate,guanidine, e.g., arginine, hydroxyl, e.g., serine and threonine, andthiol, e.g., cysteine. Pendant groups may also be employed to providethe desired functionalities. For example, carboxy groups may be reactedwith polyamines so as to exchange a carboxyl functionality for a singleamino or plurality of amino groups. An amino group may be substitutedwith a polycarboxylic acid, so that the amino group will be replacedwith a plurality of carboxylic acid groups. A thiol may be replaced withan aldehyde, by reaction with an aldehydic olefin, e.g. acrolein, so asto provide for an aldehyde functionality. Other functionalities whichmay be introduced for crosslinking purposes may include, for example,phosphate esters, activated olefins, e.g., maleimido, thioisocyanato,and the like as well as other reactive functionalities which are knownin the art. By appropriate choice of the reactive functionalities on theprotein and the crosslinking agent (see below), the rate of reaction andthe degree of crosslinking can be controlled.

In order to crosslink the above described proteins for the purpose offorming an adhesive matrix, various chemical and/or enzymaticcrosslinkers which have been used previously and have been found to bephysiologically acceptable may find use. Various reactivefunctionalities may be employed, such as aldehyde, isocyanate, mixedcarboxylic acid anhydride, e.g. ethoxycarbonyl anhydride, activatedolefin, activated halo, amino, and the like. Preferred chemicalcrosslinkers include dialdehydes, such as glutaraldehyde, formaldehyde,activated diolefins, diisocyanates such as tetramethylene diisocyanate,hexamethylene diisocyanate, octamethylene diisocyanate,1,6-diisocyanatohexane (HMDI),4-isocyanatomethylphenyl-3-isocyanatopropanate (IMP), acid anhydrides,such as succinic acid anhydride, ethylene diamine tetraacetic aciddianhydride, diamines, such as hexamethylene diamine,cyclo(L-lysyl-L-lysine), L-lysine, and the like. The crosslinking agentmay be a free radical, initiating vinyl polymerization, such as amixture of a tertiary amine and eosin B, which is light activated, atertiary amine and a persulfate, which is thermally activated, and thelike. The crosslinking agent may also contain unsymmetricalfunctionalities, for example, activated olefin aldehydes, e.g., acroleinand quinoid aldehydes, activated halocarboxylic acid anhydride, and thelike. The crosslinking agents will usually be commercially available ormay be readily synthesized in accordance with conventional ways, eitherprior to application of the adhesive or sealant or by synthesis in situ.

Enzymatic crosslinkers may also find use in the presently describedmethods. Examples of such enzymatic-crosslinkers which find use (in thepresent invention include, for example, tyrosine oxidase, lysyl oxidase,phosphorylases, such as cellular phosphorylase A or B, glycosylases, andfatty acyltransferases. Transamidases are preferred, particularlytransglutaminases, specifically liver, muscle, epithelial orkeratinocyte transglutaminases and Factor XIII.

Thus, as used herein, the phrase “tissue adhesive” refers to acomposition which may be employed independently to bond tissuestogether. The phrase “tissue sealant” refers to the same or differentcomposition or formulation which may be employed to seal defects intissues, created surgically or otherwise. The use of adhesive substratesand crosslinkers to prepare a tissue adhesive or sealant for use in thepresently claimed invention is known in the art. Moreover, tissueadhesives and sealants are known in the art, all of which areencompassed for use in the present invention.

The methods of the present invention may be practiced by applying thetissue adhesive or sealant and the primer molecule to the tissue ofinterest wherein the tissue adhesive or sealant and primer molecule arecombined prior to applying the mixture to the tissue. Alternatively, theprimer molecule or the tissue adhesive or sealant may be applied to thetissue followed by subsequent application of the tissue adhesive orsealant or primer molecule, respectively. In a preferred embodiment, theprimer molecule first applied to the tissue, thereby allowing the primermolecule to interact with one or more proteins present in the tissue,followed by subsequent application of the tissue adhesive or sealant.The crosslinking agent may be applied in combination with the othercomponents of the tissue adhesive or sealant or may be appliedindependently.

The tissue adhesive or sealant components and the primer molecules maybe applied to the tissue in any convenient way, for example by using asyringe, catheter, cannula, manually applying the compositions,spraying, or the like. If used as an adhesive, the compositions may beapplied prior to or after the time that the tissue segments are broughtinto proximate relationship.

Further details of the invention are illustrated in the followingnon-limiting examples.

Materials and Methods

1. Recombinant Preparation of Repetitive Unit Protein Polymers

The construction of large synthetic DNA and its use in the recombinantpreparation of a variety of different repetitive unit protein polymershas been described in U.S. Pat. No. 5,243,038, PCT/US89/05016,PCT/US92/09485 and PCT/US96/06229, the disclosures of which areexpressly incorporated herein by reference in their entirety.

2. Preparation of Test Coupons

A. Preparation of Rat Skin Test Coupons [CP1]

Hides were collected from freshly sacrificed (250-300 g) femaleSprague-Dawley rats. Freshly harvested hides were interleaved betweenlayers of gauze soaked in phosphate buffered saline (PBS), placed in asealed plastic bag, and stored in a freezer at −20° C. Hides were thawedapproximately 30-60 minutes prior to use. Thawing was accomplished byusing natural convection at room temperature or by placing the sealedbag of hides in a water bath at 40° C. Thawed hides were cut with astandard single-edged razor blade into 1 cm×3 cm coupons, and thenaggressively scraped with a razor blade to remove all fascia and loosemuscle.

Coupons were interleaved between gauze soaked in PBS and placed insealed plastic bags. The sealed bags were either placed in an ice bathfor short term storage or were placed in a −20° C. freezer for long termstorage. Thawing prior to use was accomplished as described above forwhole hides.

B. Preparation of Myocardium Test Coupons [CP2]

Fresh bovine hearts were obtained from an abattoir, rinsed with PBS, andstored frozen until use. The frozen heart was allowed to partially thawat room temperature, and was cut into slabs about 2 mm thick using astandard meat slicer. Slabs were cut with a standard single edged razorblade into 1 cm×3 cm coupons, interleaved between gauze soaked in PBS,and placed in sealed plastic bags. The sealed bag was placed in an icebath for short term storage. No long term storage of myocardium couponswas attempted. Myocardium displays a pronounced direction to thealignment of the muscle fibers. Test coupons were cut relative to thisalignment: [CP2p] longitudinal coupons were cut with the long axis ofthe coupon parallel to the muscle fiber axis; [CP2t] transverse couponswere cut with the long axis of the coupon perpendicular to the musclefiber axis; [CP2d] diagonal coupons were cut with the long axis of thecoupon at 45° to the muscle fiber axis.

C. Bone Test Coupons [CP3]

Fresh bovine knee joints were obtained from an abattoir, rinsed withPBS, and stored refrigerated until use. Slabs of cancellous bone 2 mmthick were cut from the head of the femur using a fine toothed saw. Theslabs were trimmed into coupons 1 cm×2 cm. One end of each coupon, ca. 1cm×1 cm, was reduced in thickness to about 1 mm using a file. Bone testcoupons were interleaved between gauze soaked in PBS, and placed insealed plastic bags. The sealed bag was placed in an ice bath for shortterm storage. No long term storage of bone coupons was attempted. Thestepped shape described for these test coupons permitted the tensilepull in the Instron testing to occur in line with the axis of the lapjoint.

D. Tendon Test Coupons [CP4]

Fresh bovine knee joints were obtained from an abattoir, rinsed withPBS, and stored refrigerated until use. Tendon was dissected from thejoint and trimmed into coupons 2 cm long×0.8 cm wide×0.1 cm thick withthe long axis of the coupon parallel to the long axis of the tendon.Tendon test coupons were interleaved between gauze soaked in PBS, andplaced in sealed plastic bags. The sealed bag was placed in an ice bathfor short term storage. No long term storage of tendon, coupons wasattempted.

E. Articular Cartilage Test Coupons [CP5]

Fresh bovine knee joints were obtained from an abattoir, rinsed withPBS, and stored refrigerated until use. Articular cartilage wasdissected from the joint and trimmed into coupons 2 cm long×1 cmwide×0.05 cm thick. Articular cartilage test coupons were interleavedbetween gauze soaked in PBS, and placed in sealed plastic bags. Thesealed bag was placed in an ice bath for short term storage. No longterm storage of articular cartilage coupons was attempted.

The mechanics of a lap shear tensile test are such that the maximumstrength observed with a given adhesive is dependent upon the mechanicalcompliance of the adherend coupons used in the test, with stiffercoupons exhibiting a greater apparent strength to failure. (Kendall,“Adhesion 2,” Edited by K. Allen, Applied Science Publishers, London, p.121, (1978) and Kendall, Journal of Physics, D: Applied Physics, 8:512(1975)). Consequently,.coupons for a related set of tests were selectedto be as consistent as possible, especially with respect to thickness.Minimizing variance in the compliance of the test coupons serves tominimize the coefficient of variation observed in a given set of tests,thereby making it possible to discern effects due to changes in theperformance of the adhesive.

3. Estimation of Liquid Densities

About 1.0 ml of liquid to be tested was drawn into 1.00 mL tuberculinsyringe, the syringe inverted, and the plunger depressed to eject allliquid from the syringe, leaving the Luer hub and needle full of liquid(no air bubbles), and the wetted assembly tared. The syringe was filledto the 1.00 mL mark with the liquid to be tested, the gross weight ofthe filled syringe was measured, the net weight of the 1.00 mL incrementof liquid was calculated, and the density of the liquid was recorded ing/mL. This process was repeated five times, and the mean of these valueswas used as the estimate of the density of the liquid. Densities of dopesolutions and crosslinkers are used in calculations of stoichiometriesin several of the adhesive systems and application techniques describedbelow.

4. Crosslinking Agents

The following crosslinking agents were employed:

Glutaraldehyde [GA]: Glutaraldehyde (50% w/w, Fisher G151-1) wasdistilled and diluted with deionized water to a concentration of 25%(w/w), and stored at 4° C. (Whipple et al., Journal of OrganicChemistry, 39(12):l666-1669 (1974) and Hardy et al., Journal of theChemical Society, Chemical Communications, pp. 565-566 (1969)). Thedensity of this solution was estimated as 1.058 g/mL.

Formaldehyde [FA]: Formaldehyde (37% w/w, Aldrich 25,254-9) was used asreceived. The density of this solution was estimated as 1.110 g/mL

1,6-Diisocyanatohexane [HMDI]: 1,6-Diisocyanatohexane (Aldrich,D12,470-2) was used as received. Product literature specifies itsdensity as 1.040 g/mL.

4-Isocyanatomethylphenyl-3-isocyantopropanate [IMP]: To a mixture of4-hydroxy-phenylacetic acid (76 g, 0.50 mole) and succinic anhydride (50g, 0.50 mole) in chloroform (400 mL), triethylamine (101.2 g, 1.00 mole)was added dropwise at a rate to control the exotherm.

After the initial exothermic reaction subsided, the mixture was refluxedfor 2 hours; FTIR (CHCl₃)1759 cm⁻¹, 1598 cm⁻¹. The reaction mixture wascooled in an ice bath and thionyl chloride (136.8 g, 1.15 mole) wasadded dropwise over 90 minutes. The reaction mixture was stirred 30minutes without heating and then refluxed for 2 hours. Solvents wereevaporated at reduced pressure using a rotary evaporator. Toluene (150mL) was added and the white precipitate was filtered, giving a lightbrown clear solution. This solution was diluted with additional toluene(100 mL) and added dropwise over 4 hours to a mixture of sodium azide(65 g, 1.0 mole) in toluene (50 mL) heated in an oil bath at 120° C.Heating at 120° C. was continued for an additional 4 hours. The mixturewas cooled to room temperature and filtered. Toluene was removed fromthe filtrate by evaporation on the vacuum line. The residue wassubjected to distillation at reduced pressure using a wide bore shortpath distillation head. Product distilled as a yellow—amber viscousliquid (6.09 g, 0.025 mole,. 5.0%) between 100°@ 20 mTorr and 120°@ 35mTorr. ¹H nmr (CDCl₃) δ 7.34(d,2H), 7.12 (d, 2H), 4.50 (s, 2H), 3.71(t,2H), 2.87 (t,2H); FTIR (neat) v_(NCO) 2272 cm⁻¹, v_(C═O) 1760 cm⁻¹.The density this product was estimated as 1.236 g/mL.

The dimethylamine adduct was formed by adding dimethylamine (1.0 mL of2.0 M solution in THF) to 4-isocyanatomethylphenyl-3-isocyantopropanate(0.25 g, 1.0.mmole) in anhydrous diethyl ether (1.0 mL) cooled in an icebath. The mixture was stirred for 30minutes in the ice bath and for 30minutes at room temperature. Anhydrous diethyl ether (5.0 mL) was added,stirred 5 minutes, and the white precipitate filtered, washed withdiethyl ether, and dried in vacuum. FTIR (KBr) 3325 cm⁻¹, 1754 cm⁻¹,1635 cm⁻¹; ES MS m/z=337 (M−H⁺).

Factor XIIIa [F XIIIa]: Factor XIIIa was prepared as in [PD6] (seebelow) from the TISSEEL® Kit VH, 2-Component Fibrin Sealant (HUMAN),Vapor Heated, 1.0 mL. [IMMUNO (Canada) Ltd., Catalog No. P199859506S]

5. Preparation of Dopes

Gelatin-Resorcinol-Glutaraldehyde (GRG) Dope [PD1]: Gelatin (200 mg,type B, 225 bloom, Sigma, Cat. No. G9382) and resorcinol (20 mg, 30Aldrich 30,752-1) were placed in a 100 mm×12 mm test tube and vortexedwith 2.0 mL of deionized water until dissolved. The use of a GRG dope isreported by Johnson, U.S. Pat. No. 5,292,333, issued on Mar. 8, 1994.

Gelatin-Resorcinol-Formaldehyde (GRF) Dope [PD2]: Resorcinol (13.5 g,Aldrich 30,752-1) and 43.5 mL of deionized water were placed into around bottom flask fitted with a mechanical stirrer and heated to 45° C.in a water bath. Gelatin (40.5 g, type B. 225 bloom, Sigma, Cat. No.G9382) was added in portions over a 4 hour period, and the mixturestirred for an additional 16 hours. The use of a GRF dope is reported byVandor et al., Zeit. Exper. Chirurg., 13: 43-51.(1980) and Koehnlein etal., Surgery, 66:377-382 (1966).

SELP8K Dope [PD3]: Protein polymer SELP8K (100 mg) and 1.0 mL ofdeionized water was placed in an 2.0 mL Eppendorf® tube and vortexeduntil dissolved. Using a 2 mm glass rod, an aliquot of this dope wasstreaked across E. Merck colorpHast® test paper, pH range 0-14. Thehydrogen ion concentration of this dope was estimated as between pH7.5 - 8.0.

SELP0K-CS1 Dope [PD4]: Protein polymer SELP0K-CS1 was used to preparethis dope as a 17% (w/w) solution in water. For example, SELP0K-CS1(2.028 g) and deionized water (9.205 g, 18 MΩ) water were vortexed in a15 mL centrifuge tube until homogeneous. Hydrochloric acid (5 μl, 3.6%w/w) was added to the dope, the solution vortexed, and then centrifugedto coalesce bubbles. The density of this dope is estimated as 1.04 g/mL.Dope [PD4] (0.20 mL) was added to deionized water (3.20 mL, 18 MΩ) toprepare a solution with a final concentration of 10.4 mg/mL, and thehydrogen ion concentration was measured using a Radiometer CopenhagenPHM 93 reference pH meter. The goal was to achieve pH=8.3±0.1 for thisdiluted solution of dope. In the event the pH fell outside of thedesired range, further increments of hydrochloric acid (3.6% w/w) orsodium hydroxide (2.0% w/w) were added to the concentrated dope and thedilution and measurement process repeated.

Subsequent preparations of this dope were made with protein polymerSELP0K-CS1 and varied slightly in final pH [pH] (pH=8.37), (pH=8.29),(pH=8.20), (pH=8.26), (pH=8.26), (pH=8.28), and (pH=8.31).

SELP0K-CS1 Dope [PD5]: Protein polymer SELP0K-CS1(1.67 g) and deionizedwater (7.597 g, 18 MΩ) water were vortexed in a 15 mL centrifuge tubeuntil homogeneous. Hydrochloric acid (33 μl, 3.6% w/w) was added to thedope, the solution vortexed, and then centrifuged to coalesce bubbles.The density of this dope is estimated as 1.04 g/mL. Dope [PD5] (0.20 mL)was added to deionized water (3.20 mL, 18 MΩ) to prepare a solution witha final concentration of 10.4 mg/mL, and the hydrogen ion concentrationwas measured using a Radiometer. Copenhagen PHM 93 reference pH meter aspH=7.27.

TISSEEL® [PD6]: All operations of this procedure were performed in the37° C. warm room. The lyophilized TISSEEL® and the aprotinin solutionfrom the kit were equilibrated to 37° C. for 30 minutes before addingthe aprotinin solution to the TISSEEL®. The mixture was allowed to standfor 5 minutes, swirled briefly, and stirred on a magnetic stirrer for 15minutes. The mixture was stored at 37° C. until used. The calciumchloride solution supplied with the kit was transferred into thelyophilized Thrombin 4, swirled briefly, and stored at 37° C. until used(the preparation of these solutions was per manufacturer's instructions;TISSEEL KIT VH, 2-Component Fibrin Sealant (HUMAN), Vapor Heated, 1.0mL; IMMUNO (Canada) Ltd., Catalog #P199859506S).

SELP0K-CS1 Dope [PD7]: Protein polymer SELP0K-CS1(1.91 g) and deionizedwater (8.675 g, 18 MΩ) were vortexed in a 15 mL centrifuge tube untilhomogeneous. Hydrochloric acid (18 μl, 3.6% w/w) was added to the dope,the solution vortexed, and then centrifuged. The density of this dope isestimated as 1.04 g/mL. Dope [PD7] (0.20 mL) was added to deionizedwater (3.20 mL, 18 MΩ) to prepare a solution with a final concentrationof 10.4 mg/mL, and the hydrogen ion concentration was measured using aRadiometer Copenhagen PHM 93 reference pH meter as pH=8.30.

SELP0K-CS1 Dope [PD8]: Dope [PD7] (0.36 mL, 0.3715 g) was added bysyringe into a 2 mL cryogenic tube. Brilliant Blue G (3.7 mg, [P1]) wasadded and dissolved by vortexing for one minute.

SELP0K-CS1 Dope [PD9]: Protein polymer SELP0K-CS1 (1.6 grams) anddeionized water (7.25 grams, 18 MW) were vortexed in a 15 ml centrifugetube until homogeneous. Hydrochloric acid (55 μl, 4.4% w/w) was added tothe dope, the solution vortexed, and then centrifuged. The density ofthis dope is estimated as 1.04 g/ml. Dope [PD9] (0.2 ml) was added todeionized water (3.2 ml, 18 MW) to prepare a solution with a finalconcentration of 10.4 mg/ml, and the hydrogen ion concentration wasmeasured using a Radiometer Copenhagen PHM 93 reference pH meter aspH=8.24.

SELP0K-CS1 Dope [PD10]: Protein polymer SELP0K-CS1 (1.59 grams) anddeionized water (7.43 grams, 18 MW) were used to prepare a dope cognateto [PD9]. Dope [PD10] (0.2 ml) was added to deionized water (3.2 ml, 18MW) to prepare a solution with a final concentration of 10.4 mg/ml, andthe hydrogen ion concentration was measured using a RadiometerCopenhagen PHM 93 reference pH meter as pH=7.4.

SELP0K-CS1 Dope [PD11]: Protein polymer SELP0K-CS1 (1.49 grams) anddeionized water (6.971 grams, 18 MW) were vortexed in a 15 ml centrifugetube until homogeneous. Hydrochloric acid (42 μl, 4.4% w/w) was added tothe dope, the solution vortexed, and then centrifuged. The density ofthis dope is estimated as 1.04 g/ml. Dope [PD11] (0.2 ml) was added todeionized water (3.2 ml, 18 MW) to prepare a solution with a finalconcentration of 10.4 mg/ml, and the hydrogen ion concentration wasmeasured using a Radiometer Copenhagen PHM 93 reference pH meter aspH=8.00.

6. Preparation of Primer Solutions

[PP1]: Using a four place analytical balance, primers (10 mg±0.3 mg)were weighed into 2 mL cryogenic tubes, the required amount of 100 mMsodium chloride solution added to bring them up at 1% w/w, and themixture vortexed for 2 minutes. Solutions of primers were prepared justprior to application.

[PP2]: Using a four place analytical balance, primers (10 mg±0.3 mg)were weighed into 2 mL cryogenic tubes, the required amount of 100 mMsodium chloride solution added to bring them up at 5% w/w, and themixture vortexed for 2 minutes. Solutions of primers were prepared justprior to application.

[PP3]: Protein polymer SELP0K-CS1 dope [PD4] (pH=8.28) was used asprimer.

[PP4]: Protein polymer SELP0K-CS1 dope [PD4] (0.40 mL) was diluted withdeionized water (0.28 mL, 18 MΩ) to prepare a 10% w/v solution.

[PP5]: Primer solution [PP4] (0.20 mL) was diluted with deionized water(0.20 mL, 18 MΩ) to prepare a 5% w/v solution.

[PP6]: Primer solution [PP4] (0.10 mL) was diluted with deionized water(0.40 mL, 18 MΩ) to prepare a 1% w/v solution.

[PP7]: Approximately 2M aqueous urea was prepared as a primer. Urea (60g, 1.00 mole) was added to a 100 mL volumetric flask and deionized water(18 MΩ) was added to the mark, and the mixture agitated until dissolvedto yield a 10 M stock solution. An aliquot (4.00 mL) of 10 M stock wasdiluted with deionized water (6.00 mL, 18 MΩ) and adjusted to pH=7.2 bythe addition of about 30 μL of concentrated hydrochloric acid to yieldan approximately 4M stock solution. An aliquot of 4M stock (0.50 mL) wasdiluted with deionized water (0.50 mL, 18 MΩ) to yield an approximately2M solution.

[PP8]: Tisseel® was dissolved in a solution of aprotinin as prepared in[PD6].

[PP9]: Primer [P1; see below] was added directly into the adhesive dopeas described in the preparation of [PD8]. No separate priming step wasused.

[PP10]: Using a four place analytical balance, primers (100 mg±1 mg)were weighed into 2 ml cryogenic tubes, the required amount of 100 mMsodium chloride solution added to bring them up to 10% w/w, and themixture vortexed for 2 minutes. Solutions of the primers were preparedjust prior to application.

7. Identification of Primer Molecules

A key of primer molecules, their color index number (for the dyes),manufacturer, and catalog number follows: [P1]: Brilliant Blue G (CI#42655) Aldrich 20,140-5 [P2]: Evan's Blue (CI# 23860) Aldrich 20,633-4[P3]: Chicago Sky Blue 6B (CI# 24410) Aldrich 20,138-3 [P4]: CibacronBlue 3GA (CA# 12236-82-7) Sigma C9534 [P5]: Cibacron Brilliant Yellow3G-P (CI# 18972) Aldrich 22,847-8 [P6]: Brilliant Blue R (CI#42660)Aldrich 20,140-5 [P7]: Lissamine Green B (CI# 44090) Aldrich 19,958-3[P8]: Acid Blue 92 (CI# 13390) Aldrich 21,042-0 [P9]: Cibacron BrilliantRed 3B-A (CI# 18105) Aldrich 22,845-1 [P10]: Acid Red 97 (CI# 22890)Aldrich 21,039-0 [P11]: Trypan Blue (CI# 23850) Aldrich 30,264-3 [P12]:New Coccine (CI#16255) Aldrich 19,973-7 [P18]: Orange G (CI# 16230)Aldrich 86,128-6 [P19]: Urea Aldrich 20,888-4 [P20]: Tisseel ®Fibrinogen IMMUNO, Ltd., # P199859506S [P21]: SELP0K-CS1 n/a [P22]:Eosin Y (CI# 45380) Aldrich 11,983-0 [P23]: Eosin B (CI# 45400) Aldrich86,100-6 [P24]: Erythrosin B (CI# 45430) Aldrich 19,826-9 [P25]: RoseBengal (CI# 45440) Aldrich 19,825-0

8. Application of Primer Molecules to Test Coupons

[AP1]: All operations of this procedure were performed in the 37° C.warm room. Test coupons were placed on a glass plate and covered with asheet of plastic film while they equilibrated to 37° C. Primer solution(5 μL) was applied to each coupon of the adhesive joint using aP-20.Pipetman®. Care was taken to distribute the primer solutionuniformly over the 1 cm×1 cm area intended for the joint. The primersolution was allowed to set on the coupon for 1-2 minutes before theexcess was blotted off using an adsorbent tissue paper.

[AP2]: Cognate to [AP1] except 10 μl of primer solution was applied percoupon.

[AP3]: All operations of this procedure were conducted at ambient roomtemperature of approximately 23° C. Test coupons were placed on a glassplate and covered with a sheet of plastic film while they equilibratedto room temperature. Primer solution (5 μL) was applied to each couponof the adhesive joint using a P-20 Pipetman®. Care was taken todistribute the primer solution uniformly over the 1 cm×1 cm areaintended for the joint. The primer solution was allowed to set on thecoupon for 2-3 minutes before the excess was blotted off using anadsorbent tissue paper.

[AP4]: All operations of this procedure were conducted in a 37° C. warmroom. Test coupons were placed on a glass plate and covered with a sheetof plastic film while they equilibrated to 37° C. Primer solution (5 μL)was applied to each coupon of the adhesive joint using a P-20 Pipetman®.Care was taken to distribute the primer solution uniformly over the 1cm×1 cm area intended for the joint. The primer solution was allowed toset on the coupon for 2-3 minutes before the excess was blotted offusing an adsorbent tissue paper.

[AP5]: All operations of this procedure were conducted in a 37° C. warmroom. Test coupons were placed on a glass plate and covered with plasticfilm while they equilibrated to 37° C. Primer solution (15 μL) wasapplied to each coupon of the adhesive joint using a P-20 Pipetman®.Care was taken to distribute the primer solution uniformly over the 1cm×1 cm area intended for the joint. The primer solution wasaggressively worked into the surface of each coupon using 30-40 strokeswith a flat bladed stainless steel spatula.

[AP6]: Cognate to [AP5] except 5 μl of primer solution was applied percoupon.

[AP7]: Primer was added directly to the dope preparation [PD8]. Noseparate application of primer was used.

[AP8]: Cognate to [AP3] except that the primer was allowed to set for5-10 minutes, and excess primer was not blotted off with an absorbenttissue paper.

[AP9]: Cognate to [AP8] except that 7 μl of primer was applied to eachcoupon of the adhesive joint.

9. Application of Dopes

Premixed Compositions of Dope Plus Crosslinker

Those compositions of dope plus crosslinker referred to below as being“premixed” were prepared using a dynamic mixing apparatus consisting ofa 0.0625″ diameter steel shaft supported at the proximal end in acompression bushing and rotating at 10,000 rpm in a Teflon® body boredto 0.078″ diameter and open at the distal end. Dope and crosslinker werefed to the mixing apparatus using individual syringe pumps so that thefeedrate of each component could be independently controlled. Eachcomponent entered the mixing chamber through it's own port located nearthe proximal end. The active length of the mixing chamber distal to theinlet ports was 1.28″. The output of the mixing apparatus was applieddirectly to the 1 cm×1 cm surfaces of the intended joint on the testcoupons.

GRG system [AD1]: All operations of this procedure were conducted atambient room temperature of approximately 23° C. Two test coupons and amicroscope slide were placed on a glass plate, the coupons covered witha sheet of plastic film to prevent evaporation of moisture, equilibratedto room temperature, and the assembly tared. Glutaraldehyde (2 μL, 25%w/w) was distributed evenly over the 1cm×1 cm area of the intendedadhesive joint on one test coupon using a P-20 Pipetman®. GRG dope [PD1](10 μL) was applied evenly over the glutaraldehyde on the first couponusing a P-20 Pipetman®. The joint was assembled and then covered withthe sheet of plastic film to retard evaporation of moisture. Themicroscope slide was placed on top of the plastic film and the joint.This whole assembly was placed onto a balance and a force of 2000±250 gwas applied to the joint by hand for one minute. A 100 g weight wasplaced on top of the microscope slide.

GRF system [AD2]: All operations of this procedure were conducted in a37° C. warm room. Two test coupons and a microscope slide were placed ona glass plate, the coupons covered with a sheet of plastic film toprevent evaporation of moisture, equilibrated to 37° C., and theassembly tared. GRF dope [PD2] (^(˜)10 μL) was troweled onto the 1 cm×1cm area of the intended joint on one test coupon using a flat bladedstainless steel spatula. Formaldehyde (1 μL, 37% w/w) was uniformlydistributed over the GRF dope on the first coupon using a P-20Pipetman®. The joint was assembled and then covered with the sheet ofplastic film to retard evaporation of moisture. The microscope slide wasplaced on top of the plastic film and the joint. This whole assembly wasplaced onto a balance and a force of 1500±250 g was applied to the jointby hand for 20 seconds. A 100 g weight was placed on top of themicroscope slide.

SELP8K/glutaraldehyde system [AD3]: All operations of this procedurewere conducted at ambient room temperature of approximately 23° C. Twotest coupons and a microscope slide were placed on a glass plate, thecoupons covered with a sheet of plastic film to prevent evaporation ofmoisture, equilibrated to room temperature, and the assembly tared.SELP8K dope [PD3] (5 μL per coupon) was uniformly distributed using aP-20 Pipetman® over the 1 cm×1 cm area intended for the joint.Glutaraldehyde (3.3 μL, 1.0 M) was uniformly distributed over the dopeon one coupon. The joint was assembled and then covered with the sheetof plastic film to retard evaporation of moisture. The microscope slidewas placed on top of the plastic film and the joint. A 100 g weight wasplaced on top of the microscope slide.

SELP0K-CS1 [PD4] system [AD4]: All the operations of this procedure wereconducted at ambient room temperature of approximately 23° C. The dopeand crosslinker were premixed. The flowrate of [PD4] dope was 22.54ml/hr, and the flowrate of the HMDI was 0.67 ml/hr. The ratio ofisocyanate groups from the HMDI to the amino groups from the [PD4] dopewas approximately 10:1. Two test coupons and a microscope slide wereplaced on a glass plate, the coupons covered with a sheet of plasticfilm to prevent evaporation of moisture, equilibrated to roomtemperature, and the assembly tared. A hemispherical droplet, about 5 mmin diameter, of the premixed components was applied to each of the twotest coupons. A gentle troweling motion with a flat bladed stainlesssteel spatula was used to uniformly distribute the droplet over the 1cm×1 cm area intended for the joint. The joint was assembled and thencovered with the sheet of plastic film to retard evaporation ofmoisture. The microscope slide was placed on top of the plastic film andthe joint. This whole assembly was placed onto a tared balance and aforce of 1500±250 g was applied to the joint by hand for one minute. A100 g weight was place on top of the microscope slide.

SELP0K-CS1 [PD5] system [AD5]: Cognate to [AD4] except the flowrate of[PD5] dope was 22.39 mL/hr, and the flowrate of the IMP was 0.83 mL/hr.The ratio of isocyanate groups from the IMP to the amino groups from the[PD5] dope was approximately 10:1.

TISSEEL [PD6] system [AD6]: All of the operations of this procedure wereconducted in a 37° C. warm room. Two test coupons and a microscope slidewere placed on a glass plate, the coupons covered with a sheet ofplastic film to prevent evaporation of moisture, equilibrated to 37° C.,and the assembly tared. A P-20 Pipetman® was used to apply the TISSEELsolution [PD6] (20 μL) to one coupon and the Thrombin 4 solution (20 μL)to the second coupon. Care was taken to uniformly distributed thesolutions across the 1 cm×1 cm area intended for the joint. The jointwas assembled and then covered with the sheet of plastic film to retardevaporation of moisture. The microscope slide was placed on top of theplastic film and the joint. This whole assembly was placed onto abalance and a force of 1500±250 g was applied to the joint by hand forone minute. A 100 g weight was then placed on top of the microscopeslide.

SELP0K-CS1 [PD7] system [AD7]: All of the operations of this procedurewere conducted in a 37° C. warm room. Two test coupons and a microscopeslide were placed on a glass plate, the coupons covered with a sheet ofplastic film to prevent evaporation of moisture, equilibrated to 37° C.,and the assembly tared. SELP0K-CS1 dope [PD7] (15 μL) was applied usinga P-20 Pipetman® to each coupon over the 1 cm×1 cm area intended for thejoint, for a total of 30 μL. The dope was aggressively worked into thesurface of the coupons using approximately 30 to 40 strokes with a flatbladed stainless steel spatula. IMP (2 μL) was then applied to both thecoupons using a P-20 Pipetman® in a pattern of streaks: three equallyspaced parallel streaks on one coupon and an “X”-shaped pattern of twostreaks on the second coupon. The approximate mole ratio of isocyanategroups form the IMP to the amino groups from the SELP0K-CS1 was 18:1.The joint was assembled and covered with the sheet of plastic film toretard the evaporation of moisture. The microscope slide was placed ontop of the plastic film and the joint. This whole assembly was placedonto a balance and a force of 1500±250 g was applied to the joint byhand for one minute. A 100 g weight was then placed on top of themicroscope slide.

SELP0K-CS1 [PD8] system [AD8]: Cognate to [AD7], except SELP0K-CS1 dope[PD8] was used.

SELP0K-CS1 [PD4] system [AD9]: Cognate to [AD4], except the premix wasaggressively worked into the surface of the coupons using approximately30 to 40 strokes with a flat bladed stainless steel spatula.

SELP0K-CS1 [PD5] system [AD10]:l Cognate to [AD5]; except the flowratesfor the SELP0K-CS1 dope [PD5] was 22.72 mL/hr and the flowrate for theIMP was 0.50 mL/hr. The ratio of isocyanate groups from the IMP to theamino groups from the [PD5] dope was approximately 6:1.

SELP0K-CS1 [PD5] system [AD11]: Cognate to [AD5], except the flowratesfor the SELP0K-CS1 dope [PD5] was 22.97 mL/hr and the flowrate for IMPwas 0.25 mL/hr. The ratio of isocyanate groups from the IMP to the aminogroups from the [PD5] dope was approximately 3:1.

SELP0K-CS1 [PD5] system [AD12]: Cognate to [AD5], except the flowratesfor the SELP0K-CS1 dope [PD5] was 23.13 mL/hr and the flowrate for IMPwas 0.08 mL/hr. The ratio of isocyanate groups from the IMP to the aminogroups from the [PD5] dope was approximately 1:1.

SELP0K-CS1 [PD9] system [AD13]: Cognate to [AD4] except that theflowrate for the SELP0K-CS1 dope was 11.28 mL/hr and the flowrate forHMDI was 0.337 mL/hr. The ratio of isocyanate groups from the HMDI tothe amino groups from the [PD9] dope was approximately 10:1, but theresidence time in the dynamic mixing apparatus was doubled.

10. Curing Conditions

[C1]—The joint was permitted to cure at 23° C. for 30 minutes

[C2]—The joint was permitted to cure at 37° C. for 60 minutes

[C3]—The joint was permitted to cure at 37° C. for 30 minutes

[C4]—The joint was permitted to cure at 37° C. for 30 to 40 minutes

[C5]—The joint was permitted to cure at 37° C. for 15 to 20 minutes.

11. Lap Shear Tensile Testing

Unless otherwise indicated, lap shear tests were conducted by carryingout the following steps:

(1) A uniform set of test coupons were selected.

(2) The solution of primer was applied to the surfaces of the joint.

(3) The dope and crosslinker was applied to the surfaces of the joint.

(4) The lap joint was assembled and cured.

(5) The strength of the lap joint was measured on an Instron tensiletesting machine.

(6) The above steps were repeated without application of primer.

(7) The improvement of strength in the presence of primer was reportedas an enhancement factor.

The lap shear tensile testing was conducted using an Instron Mini-55tensile testing machine with pneumatic grips, using a ±500N (±0.5% offull scale) load cell, and at a crosshead speed of 25 mm/min. Testingwas conducted at ambient room temperature and humidity.

Paper mounting frames were fabricated by cutting a 1″×1″ square hole inthe center of a 2″×2″ square of standard glassine weighing paper. Thelap joint specimen was affixed across the opening in the paper frameusing adhesive tape. The dimensions of the joint were measured using aruler. The use of a mounting frame facilitated accurate alignment of thetest specimen in the pneumatic grips of the testing machine. Once thespecimen was secured in the grips, the two sides of the mounting frameparallel to the specimen were cut, the gage length and load initialized,and the test started. The test was stopped shortly after the jointfailed. The data acquisition rate was 4 samples per second. A filecontaining specimen identification, load, gage length, and duration wassaved to disk. The lap shear tensile strength was calculated by dividingthe highest load recorded for the specimen by the calculated area of thejoint. Means and standard deviations for multiple measurement werecalculated using standard methods.

Results

A. Identification of the Site of Mechanical Failure of a Tissue Bond.

In an attempt to develop novel tissue adhesive and sealant systems usingthe SELP0K-CS1 protein and isocyanate crosslinkers, techniques forincreasing the mechanical strength of bonded tissue joints wereexamined. As part of this effort, rat skin test coupons were bonded witha SELP0K-CS1/isocyanate tissue adhesive, the coupons were T-peeled toinduce partial debonding, the tissue samples were fixed, stained,microtomed and the sections mounted for histological examination.Microscopic examination of the mounted sections evidenced that themechanical failure of the glued joints occurred not within thecrosslinked adhesive matrix itself, but rather at the interface betweenthe crosslinked adhesive matrix and the skin coupon. Thus, it isbelieved that the weakest component of the bond between the tissues wasat the interface between the crosslinked adhesive matrix and one or moreproteins present in the tissue.

B. Use of Primer Molecules to Enhance the Strength of the AdhesiveMatrix/Tissue Protein Interface.

In an attempt to identify novel methods for enhancing the mechanicalperformance of tissue adhesives by increasing the strength of theadhesive matrix/tissue-associated protein interface, lap shear tensiletesting as described above was performed with a variety of differentcombinations of adhesive matrix precursors, crosslinkers, primers,methods of primer preparation, methods of applying the primers, dopes,methods of applying the dopes and curing agents. The results from theseexperiments are presented in Table 1 wherein each of the above describedvariables are indicated by their codes as presented above and the numberof experiments performed per case is shown in parentheses. TABLE IPrimer ID [P#] Prep Dope [PD#] Test Adhesive Matrix Prep Primer [PP#]Appl'n Dope [AD#] Lap Shear Tensile Case Coupon Precursor CrosslinkerAppl'n Primer [AP#] Cure [C#] x ± σ (n) [g/cm²] Enhancement 1 [CP1]Gelatin GA none [PD1], [AD1], [C1]  296 ± 65 (15) — 2 [CP1] Gelatin GA[P10], [PP1], [AP3] [PD1], [AD1], [C1]  440 ± 52 (4) 1.49 X 3 [CP1]Gelatin FA none [PD2], [AD2], [C3]  670 ± 138 (6) — 4 [CP1] Gelatin FA[P10], [PP1], [AP4] [PD2], [AD2], [C3]  953 ± 235 (5) 1.42 X 5 [CP1]SELP8K GA none [PD3], [AD3], [C2]  143 ± 40 (6) — 6 [CP1] SELP8K GA[P10], [PP1], [AP3] [PD3], [AD3], [C2]  393 ± 89 (6) 2.75 X 7 [CP1]SELP8K GA [P2], [PP1], [AP3] [PD3], [AD3], [C2]  272 ± 73 (6) 1.90 X 8[CP1] SELP8K GA [P11], [PP1], [AP3] [PD3], [AD3], [C2]  242 (1) 1.69 X 9[CP1] SELP8K GA [P4], [PP1], [AP3] [PD3], [AD3], [C2]  229 (1) 1.60 X 10[CP1] SELP8K GA [P12], [PP1], [AP3] [PD3], [AD3], [C2]  225 (1) 1.57 X11 [CP1] SELP0K-CS1 HMDI none [PD4], [AD4], [C4]  452 ± 99 (12) — 12[CP1] SELP0K-CS1 HMDI [P1], [PP1], [AP1] [PD4], [AD4], [C4] 1581 ± 157(9) 3.50 X 13 [CP1] SELP0K-CS1 HMDI [P2], [PP1], [AP1] [PD4], [AD4],[C4]  972 (1) 2.15 X 14 [CP1] SELP0K-CS1 HMDI [P3], [PP1], [AP1] [PD4],[AD4], [C4]  958 (1) 2.12 X 15 [CP1] SELP0K-CS1 HMDI [P6], [PP1], [AP1][PD4], [AD4], [C4] 1153 (1) 2.55 X 16 [CP1] SELP0K-CS1 IMP none [PD5],[AD5], [C4]  422 (1) — 17 [CP1] SELP0K-CS1 IMP [P1], [PP1], [AP1] [PD5],[AD5], [C4] 2029 (1) 4.81 X 18 [CP1] SELP0K-CS1 IMP none [PD5], [AD10],[C4]  421 (1) — 19 [CP1] SELP0K-CS1 IMP [P1], [PP1], [AP1] [PD5],[AD10], [C4] 2353 (1) 5.59 X 20 [CP1] SELP0K-CS1 IMP none [PD5], [AD11],[C4]  463 (1) — 21 [CP1] SELP0K-CS1 IMP [P1], [PP1], [AP1] [PD5],[AD11], [C4] 2013 (1) 4.35 X 22 [CP1] SELP0K-CS1 IMP none [PD5], [AD12],[C4]  274 (1) — 23 [CP1] SELP0K-CS1 IMP [P1], [PP1], [AP1] [PD5],[AD12], [C4] 1241 (1) 4.53 X 24 [CP1] Fibrin F XIIIa none [PD6], [AD6],[C4]  264 ± 34 (3) — 25 [CP1] Fibrin F XIIIa [P4], [PP1], [AP1] [PD6],[AD6], [C4]  954 ± 40 (2) 3.61 X 26 [CP1] Fibrin F XIIIa [P5], [PP1],[AP1] [PD6], [AD6], [C4]  818 ± 198 (2) 3.10 X 27 [CP1] Fibrin F XIIIa[P7], [PP1], [AP1] [PD6], [AD6], [C4] 1217 ± 57 (3) 4.61 X 28 [CP1]Fibrin F XIIIa [P8], [PP1], [AP1] [PD6], [AD6], [C4] 1117 ± 337 (3) 4.21X 29 [CP1] Fibrin F XIIIa [P9], [PP1], [AP1] [PD6], [AD6], [C4]  717 ± 7(2) 2.72 X 30 [CP1] SELP0K-CS1 IMP none [PD7], [AD7], [C5] 1772 ± 394(5) — 31 [CP1] SELP0K-CS1 IMP [P1], [PP9], [AP7] [PD8], [AD8], [C5] 2699± 362 (5) 1.52 X 32 [CP1] SELP0K-CS1 HMDI none [PD4], [AD9], [C4]  763 ±95 (3) — 33 [CP1] SELP0K-CS1 HMDI [P21], [PP3], [AP5] [PD4], [AD9], [C4]1440 ± 41 (2) 1.89 X 34 [CP1] SELP0K-CS1 HMDI [P21], [PP4], [AP5] [PD4],[AD9], [C4] 1505 ± 382 (2) 1.97 X 35 [CP1] SELP0K-CS1 HMDI [P21], [PP5],[AP5] [PD4], [AD9], [C4] 1577 (1) 2.07 X 36 [CP1] SELP0K-CS1 HMDI [P21],[PP6], [AP5] [PD4], [AD9], [C4] 1055 ± 8 (2) 1.38 X 37 [CP1] SELP0K-CS1HMDI [P19], [PP7], [AP6] [PD4], [AD9], [C4] 1023 ± 61 (2) 1.34 X 38[CP1] SELP0K-CS1 HMDI [P20], [PP8], [AP6] [PD4], [AD9], [C4] 1130 ± 431(8) 1.48 X 39 [CP1] SELP0K-CS1 HMDI [P18], [PP2], [AP6] [PD4], [AD9],[C4] 1398 (1) 1.83 X 40 [CP1] SELP0K-CS1 HMDI [P5], [PP2], [AP6] [PD4],[AD9], [C4] 1523 (1) 2.00 X 41 [CP1] SELP0K-CS1 HMDI [P9], [PP2], [AP6][PD4], [AD9], [C4] 1675 (1) 2.20 X 42 [CP1] SELP0K-CS1 HMDI [P4], [PP2],[AP6] [PD4], [AD9], [C4] 1524 (1) 2.00 X 43 [CP1] SELP0K-CS1 HMDI [P1],[PP1], [AP2] [PD4], [AD9], [C4] 1821 (1) 2.39 X 44 [CP1] SELP0K-CS1 HMDI[P10], [PP1], [AP2] [PD4], [AD9], [C4] 1396 (1) 1.82 X 45 [CP2t]SELP0K-CS1 HMDI none [PD9], [AD13], [C4]  54 (1) — 46 [CP2t] SELP0K-CS1HMDI [P22], [PP10], [AP8] [PD9], [AD13], [C4]  225 ± 21 (2) 4.17 X 47[CP2p] SELP0K-CS1 HMDI none [PD9], [AD13], [C4]  32 (1) — 48 [CP2p]SELP0K-CS1 HMDI [P22], [PP10], [AP8] [PD9], [AD13], [C4]  117 ± 9 (2)3.34 X^(a) 49 [CP2d] SELP0K-CS1 HMDI none [PD11], [AD13], [C4]  33 (1) —50 [CP2d] SELP0K-CS1 HMDI [P23], [PP1], [AP9] [PD11], [AD13], [C4]  78(1) 2.36 X 51 [CP2d] SELP0K-CS1 HMDI [P24], [PP1], [AP9] [PD11], [AD13],[C4]  222 (1) 6.73 X 52 [CP2d] SELP0K-CS1 HMDI [P25], [PP1], [AP9][PD11], [AD13], [C4]  45 (1) 1.36 X 53 [CP3] SELP0K-CS1 HMDI none[PD10], [AD13], [C4]  <60 (2) — 54 [CP3] SELP0K-CS1 HMDI [P1], [PP1],[AP8] [PD10], [AD13], [C4] 1492 ± 331 (2)   24 X 55 [CP4] SELP0K-CS1HMDI none [PD10], [AD13], [C4]  470 ± 41 (3) — 56 [CP4] SELP0K-CS1 HMDI[P1], [PP1], [AP8] [PD10], [AD13], [C4]  622 ± 181 (4) 1.32 X 57 [CP5]SELP0K-CS1 HMDI none [PD10], [AD13], [C4]  114 (1) — 58 [CP5] SELP0K-CS1HMDI [P1], [PP1], [AP8] [PD10], [AD13], [C4]  476 ± 36 (3) 4.18 X^(a)The tabs of the lap joint failed under load before the joint itself.Therefore, this is a minimum enhancement.

The results presented in Table 1 demonstrate that a variety of differentprimer molecules which are capable of interacting with and altering thephysical characteristics of protein(s) present in the test couponssignificantly enhance the mechanical performance of a variety ofdifferent tissue adhesive matrix compositions. For example, case numbers1-4 presented in Table I demonstrate that the Acid Red 97 primermolecule [P10] functions to significantly enhance the mechanicalperformance of a gelatin adhesive matrix when either a glutaraldehyde orformaldehyde crosslinker is employed. These results demonstrate thatnatural proteins (i.e., gelatin) may be employed in association with achemical crosslinker (e.g., glutaraldehyde or formaldehyde) and a primermolecule to provide for improved tissue adhesive or sealant properties.

Cases 5-10 presented in Table I demonstrate that the Evan's Blue [P2],Cibacron Blue 3GA [P4], Acid Red 97 [P10], Trypan Blue [P11] and NewCoccine [P12] primer molecules were all capable of significantlyenhancing the mechanical performance of a SELP8K tissue adhesive matrixwhich employed a glutaraldehyde crosslinker. Thus, these resultsdemonstrate that primer molecules are also useful for enhancing themechanical properties of tissue adhesives or sealants based uponrecombinantly produced proteins and chemical crosslinkers.

Cases 11-15 and 32-44 presented in Table I demonstrate that theBrilliant Blue G [P1], Evan's Blue [P2], Chicago Sky Blue 6B [P3],Cibacron Blue 3GA [P4], Cibacron Brilliant Yellow 3G-P [P5], BrilliantBlue [P6], Cibacron Brilliant Red 3B-A [P9], Acid Red 97 [P10], Orange G[P18], Urea [P19], Tisseel® Fibrinogen [P20] and SELP0K-CS1 [P21] primermolecules all provided a significant enhancement in the mechanicalperformance of a SELP0K-CS1 adhesive matrix which employed a1,6-diisocyanatohexane crosslinker. As shown in Table I, numerousdifferent elements of the experiments were varied, none of whichinhibited the ability of the primer molecules from enhancing themechanical performance of the tissue bond produced.

The use of urea as a primer molecule in case number 37 is interesting inthat it implies that one mechanism of action is related to the fact thaturea is a known chaotropic agent which is routinely used to solubilizeand denature many proteins. As such, urea swells and unravels thestructure of the native collagen protein in the tissue, thereby makingit more accessible to interact with the components of a tissue adhesiveor sealant which will crosslink to form the adhesive matrix.

Case number 38 employs fibrinogen (i.e., a protein which functions as aclotting agent) as a primer molecule which provides for a significantincrease in the mechanical performance of the tissue adhesive matrix.Fibrinogen, which has evolved to interact strongly with many differentsurfaces of the body (including collagen), may function to make thesurface of the underlying tissue collagen more physically compatiblewith the components of the adhesive matrix, e.g., more wettable by thecomponents, thereby allowing the tissue adhesive or sealant to moreefficiently interact with the collagen and, in turn, form a strongerbond.

Cases 16-23, 30 and 31 presented in Table I demonstrate that theBrilliant Blue G [P1] primer molecule is capable of providing asignificant enhancement in the mechanical performance of a SELP0K-CS1adhesive matrix which employed a4-isocyanatomethylphenyl-3-isocyanatopropanate (IMP) crosslinker.Interestingly, these results also demonstrate that not only may thetissue be pretreated with the primer molecules followed by applicationof the dope and crosslinker, but also the primer molecules may bepre-mixed with the dope and crosslinker prior to application to thetissue without adversely affecting the ability of the primer molecule toenhance the mechanical properties of the tissue bond.

Cases 24-29 presented in Table I demonstrate that the Cibacron Blue 3GA[P4], Cibacron Brilliant Yellow 3G-P [P5], Lissamine Green B [P7], AcidBlue 92 [P8] and Cibacron Brilliant Red 3B-A [P9] primer molecules areall capable of significantly enhancing the mechanical performance of afibrin-based adhesive matrix which employed a Factor XIIIa crosslinker.These results demonstrate that not only may chemical crosslinkers beemployed in association with protein adhesive matrices, but enzymaticcrosslinkers (e.g., Factor XIIIa) may be employed as well.

Finally, cases 45-58 presented in Table I demonstrate that variousprimer molecules are capable of significantly enhancing the mechanicalperformance of a SELP0K-CS1-based adhesive matrix which employed an HMDIcrosslinker on a variety of different test tissues including myocardium(a tissue which possesses abundant levels of actin and myosin), bone,tendon and articular cartilage. As such, the present invention isapplicable to enhancing the mechanical performance of a variety ofadhesive matrices on a variety of different mammalian tissues.

The foregoing description details specific methods which can be employedto practice the present invention. Having detailed such specificmethods, those skilled in the art will well enough know how to devisealternative reliable methods at arriving at the same results in usingthe fruits of the present invention. Thus, however detailed theforegoing may appear in text, it should not be construed as limiting theoverall scope thereof; rather, the ambit of the present invention is tobe determined only by the lawful construction of the appended claims.All documents cited herein are expressly incorporated herein byreference.

1. A method of adhering a tissue adhesive to a tissue, said methodcomprising: applying to said tissue (a) a primer molecule and (b) atissue adhesive, said primer molecule being capable of physicallyinteracting with a component present in said tissue and therebyrendering said tissue capable of forming a bonded interface with saidtissue adhesive. 2-25 (canceled)